Stereoviews showing the anesthetic binding site of apoferritin with A) thiopental and B) pentobarbital.

<p>The hydrogen bond between the drug and Ser-27 is shown as a black dashed line. In both images the ligands are depicted as sticks; color code: carbon, yellow; nitrogen, blue; oxygen, red; and sulfur, magenta. The electron density shown is 2<i>F</i>o-<i>F</i>c density calculated from the final refined structures, contoured at sigma level of 0.8 and carved around the ligands. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032070#pone-0032070-g002" target="_blank">Figures 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032070#pone-0032070-g003" target="_blank">3</a> were generated using PyMOL (The PyMOL Molecular Graphics System, Schrödinger, LLC).</p

Chemical structures of selected barbiturates.

<p>Chemical structures of selected barbiturates.</p

Binding, physical, and activity data for barbiturates.

<p><i>^a</i>Because of the low affinity of phenobarbital for apoferritin, it was not possible to obtain a satisfactory estimate of the molar binding enthalpy.</p><p><i>^b</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032070#pone.0032070-Franks2" target="_blank">[16]</a>.</p><p><i>^c</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032070#pone.0032070-Dingemanse1" target="_blank">[41]</a>.</p><p><i>^d</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032070#pone.0032070-Tomlin1" target="_blank">[42]</a>.</p><p><i>^e</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032070#pone.0032070-Yakushiji1" target="_blank">[43]</a>.</p><p><i>^f</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032070#pone.0032070-Rho1" target="_blank">[44]</a>.</p><p><i>^g</i>Calculated using XLOGP3 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032070#pone.0032070-Cheng1" target="_blank">[45]</a>.</p

The packing density/affinity relationship for barbiturates is offset from that of the propofol-like compounds, interpreted here as the addition of an entropic penalty offset by new enthalpic gains.

<p>Shown is the relationship between packing volume (fraction of cavity volume occupied by ligand) and affinity (dissociation constant, <i>K</i>_d) for apoferritin. The filled triangles represent a series of propofol analogs, for which a linear dependence of affinity upon packing density has been documented <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032070#pone.0032070-Vedula1" target="_blank">[21]</a>. The open circles represent the barbiturates (<i>P</i>, pentobarbital; <i>T</i>, thiopental).</p

Crystallographic data collection and refinement statistics.

<p><i>^a</i>Values in parentheses correspond to the outermost resolution shell.</p

Diverse general anesthetics utilize a common binding site in apoferritin.

<p>A) Orthogonal views of the apoferritin dimer, shown as a partially transparent molecular surface encasing a cartoon representation of the backbone. The anesthetic binding site is marked by thiopental, which is shown in a red space-filling representation. B) Stereo view showing a close-up of the anesthetic binding site, in an orientation similar to that seen in the left half of the upper panel. Four different general anesthetics are shown in ball-and-stick representations: Thiopental (yellow); propofol (magenta); isoflurane (cyan); and halothane (orange). The protein backbone is shown in blue, while selected protein side chains are shown in light gray. All four compounds, despite belonging to different chemotypes, utilize the same binding cavity, in which their positions overlap extensively.</p

A Carrier Protein Strategy Yields the Structure of Dalbavancin

Many large natural product antibiotics act by specifically binding and sequestering target molecules found on bacterial cells. We have developed a new strategy to expedite the structural analysis of such antibiotic–target complexes, in which we covalently link the target molecules to carrier proteins, and then crystallize the entire carrier–target–antibiotic complex. Using native chemical ligation, we have linked the Lys-d-Ala-d-Ala binding epitope for glycopeptide antibiotics to three different carrier proteins. We show that recognition of this peptide by multiple antibiotics is not compromised by the presence of the carrier protein partner, and use this approach to determine the first-ever crystal structure for the new therapeutic dalbavancin. We also report the first crystal structure of an asymmetric ristocetin antibiotic dimer, as well as the structure of vancomycin bound to a carrier–target fusion. The dalbavancin structure reveals an antibiotic molecule that has closed around its binding partner; it also suggests mechanisms by which the drug can enhance its half-life by binding to serum proteins, and be targeted to bacterial membranes. Notably, the carrier protein approach is not limited to peptide ligands such as Lys-d-Ala-d-Ala, but is applicable to a diverse range of targets. This strategy is likely to yield structural insights that accelerate new therapeutic development

Photoaffinity Ligand for the Inhalational Anesthetic Sevoflurane Allows Mechanistic Insight into Potassium Channel Modulation

Sevoflurane is a commonly used inhaled general anesthetic. Despite this, its mechanism of action remains largely elusive. Compared to other anesthetics, sevoflurane exhibits distinct functional activity. In particular, sevoflurane is a positive modulator of voltage-gated <i>Shaker</i>-related potassium channels (K_v1.x), which are key regulators of action potentials. Here, we report the synthesis and validation of azisevoflurane, a photoaffinity ligand for the direct identification of sevoflurane binding sites in the K_v1.2 channel. Azisevoflurane retains major sevoflurane protein binding interactions and pharmacological properties within <i>in vivo</i> models. Photoactivation of azisevoflurane induces adduction to amino acid residues that accurately reported sevoflurane protein binding sites in model proteins. Pharmacologically relevant concentrations of azisevoflurane analogously potentiated wild-type K_v1.2 and the established mutant K_v1.2 G329T. In wild-type K_v1.2 channels, azisevoflurane photolabeled Leu317 within the internal S4–S5 linker, a vital helix that couples the voltage sensor to the pore region. A residue lining the same binding cavity was photolabeled by azisevoflurane and protected by sevoflurane in the K_v1.2 G329T. Mutagenesis of Leu317 in WT K_v1.2 abolished sevoflurane voltage-dependent positive modulation. Azisevoflurane additionally photolabeled a second distinct site at Thr384 near the external selectivity filter in the K_v1.2 G329T mutant. The identified sevoflurane binding sites are located in critical regions involved in gating of K_v channels and related ion channels. Azisevoflurane has thus emerged as a new tool to discover inhaled anesthetic targets and binding sites and investigate contributions of these targets to general anesthesia